Metarhizium: jack of all trades, master of many

Abstract

The genus Metarhizium and Pochonia chlamydosporia comprise a monophyletic clade of highly abundant globally distributed fungi that can transition between long-term beneficial associations with plants to transitory pathogenic associations with frequently encountered protozoans, nematodes or insects. Some very common 'specialist generalist' species are adapted to particular soil and plant ecologies, but can overpower a wide spectrum of insects with numerous enzymes and toxins that result from extensive gene duplications made possible by loss of meiosis and associated genome defence mechanisms. These species use parasexuality instead of sex to combine beneficial mutations from separate clonal individuals into one genome (Vicar of Bray dynamics). More weakly endophytic species which kill a narrow range of insects retain sexuality to facilitate host-pathogen coevolution (Red Queen dynamics). Metarhizium species can fit into numerous environments because they are very flexible at the genetic, physiological and ecological levels, providing tractable models to address how new mechanisms for econutritional heterogeneity, host switching and virulence are acquired and relate to diverse sexual life histories and speciation. Many new molecules and functions have been discovered that underpin Metarhizium associations, and have furthered our understanding of the crucial ecology of these fungi in multiple habitats.

Keywords: Metarhizium and Pochonia chlamydosporia; insect killing (entomopathogen); parasexual and asexual life histories (Red Queen and Vicar of Bray); parasitism to insects or nematodes; plant endophyte and symbiont; sexual; virulence evolution (host switching and speciation).

Figure 1.

A phylogenomic tree with the estimated time of divergence for sequenced Metarhizium species and related fungi (solid lines). Also included (red dashed lines) are the saprophyte Metarhizium marquandii and the lizard pathogen Metarhizium viride. These have not been sequenced; their branch points on the phylogeny are estimated from a multi-gene phylogeny [32]. Right of tree, genome size, spore size and total number of genes. The number of secondary metabolite (SM) gene clusters and copy numbers of genes encoding proteases (subtilisins and trypsins) and carbohydrate-degrading glycoside hydrolases (GH), specifically GH18 (chitinases), are provided as examples of activities that likely contributed to the evolution of diverse lifestyle options. Terminal taxon names are colour coded to indicate nutritional modes (as shown in the key). Information for this figure is compiled from genome sequences described in [,,–36]. Two Pochonia chlamydosporia genomes have been sequenced (PC170 and PC123) which differ in numbers of S8 subtilisins (31 versus 25) and GH18 chitinases (18 versus 23) [33]. We arbitrarily used the numbers for PC170.

Figure 2.

Scanning electron micrograph of Metarhizium robertsii strain 2575 growing on the surface of a Manduca sexta caterpillar (a). The fungus is meandering over the cuticle until it comes across hair sockets which trigger production of small terminal appressoria; hair sockets represent a zone of weakness in the cuticle which the fungus exploits [49]. (b) Microscope image of M. robertsii 2575 growing on a fly wing, incubated with pNP-propionate to demonstrate esterase activity. (c) Diagrammatic cross section of an infected cuticle showing a germinating Metarhizium spore differentiating appressoria covered in mucus, and producing an infection hyphae that grows down into and through the cuticle (green). As shown in the key, the infectious Metarhizium produces a sequence of enzymes during penetration starting with lipases and cytochrome P450s that target epicuticular components, and then diverse promiscuous proteases that solubilize procuticular proteins to peptides that are further broken down by more specific enzymes. The redundancy of enzymes may in part be due to protease inhibitors produced by hosts to defend the cuticle (see text). (d) Blastospores and short hyphal lengths of GFP-expressing M. anisopliae strain 549 (a generalist fungal strain that does not produce destruxins) visible in the haemocoel from outside a still living fruit fly. (e) A pre-mortem squash preparation of an infected fly showing blastospores and short hyphal lengths. This particular fly line has high tolerance to fungal growth; other fly lines were much less tolerant and would not contain a high fungal load before death [50]. (f) Images of sporulating M. anisopliae and M. acridum on cockroach and grasshopper cadavers, respectively.

Figure 3.

Highly simplified overview of differentially regulated signalling pathways employed by M. robertsii 2575 infecting cockroach and locust cuticles. Host signals are sensed by G-protein coupled and other receptors. The receptors relay signals via the mitogen-activated protein kinases (MAPK) and/or the cAMP protein kinase (PKA) relays that in turn modulate the activities of transcription factors. AC, adenylate cyclase; PLC, phosphatidyl inositol-specific phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-triphosphate; DAG, diacylglycerol; PKC, protein kinase C; CaMK, calcium/calmodulin regulated kinase; ERK, extracellular signal-regulated protein kinase; CREB, cAMP response element-binding protein.

Figure 4.

Major transitions in the evolution of Metarhizium species. The lineage may have arisen from saprophytes that accumulated carbohydrate degrading enzymes (CAZymes) to degrade plant material, and presumably first became endophytes after attraction to roots by exudates. The rhizosphere provides a habitat where amoeba, nematodes, insects and fungi interact facilitating interkingdom host jumping. The close relationship of Metarhizium and P. chlamydosporia may indicate that parasitism evolved in a common ancestor. Entomopathogenicity may have evolved first, assuming asexual P. chlamydosporia evolved from sexual forms, as sexual morphs are too large for nematodes, but have been found on beetle larvae. Metarhizium genotypes with broad host ranges have been selected principally to soil and plant root habitat, not to host insect. Their population structures are clonal with parasexuality within each biotype potentially combining adaptive mutations that arise in separate lineages into one genome. The absence of sex allows extensive gene duplication that together with horizontal gene transfer (HGT) has provided generalists with a large armamentarium of enzymes and toxins able to overcome many insects. Generalist Metarhizium species and P. chlamydosporia have retained the ancestral root association, but compared to saprophytes, Metarhizium species at least have an additional ability to pass animal-derived nitrogen to the plants in exchange for carbon. Many other genotypes with evolutionary histories of insect host specificity have retained sexuality and have a larger number of rapidly evolving genes, possibly as part of an evolutionary arms race with hosts. For the most part these fungi have specialized to above-ground insects, and have reduced plant associations.

Figure 5.

Metarhizium robertsii strain 2575 expressing RFP and Trichoderma harzianum strain T12 expressing GFP growing down a grass root (a) or against a plastic surface in the presence of 0.01% (b) or 0.1% (c) yeast extract medium. (d). Metarhizium robertsii strain 2575 expressing RFP and M. majus strain 1946 expressing GFP co-inoculated onto Arabidopsis roots; strain 2575 forms a network over the root whereas 1946 shows low level germination. (e) Effects of M. robertsii inoculation on Arabidopsis root hair development. Arabidopsis was grown for 10 days on agar medium plus (+Mr2575) or minus (−Mr2575, control). RH, root hair; C, spore; H, hypha. Scale bar, 30 µm. The inset shows immunolocalization of auxin IAA (red) in the mucus secreted by a green fluorescent protein-tagged germinating M. robertsii 2575 spore (frames E and F from reference [132]).